我们感谢 Dr. 抢 Sladek 的使用喷墨观察员。我们承认加拿大卫生研究所 (研究院)、加拿大自然科学和工程研究理事会 (NSERC)、加拿大癌症协会研究所和加拿大创新基金会 (CFI) 的最后支持。感谢来自加拿大研究主席的支持。

1. 快照芯片的制作和存储 <ol> <li> 单传输方法 ( 图 1a ) <ol> <li> 现场 cAb 解决方案, 包含400和 #181; 在磷酸盐缓冲盐水中的 g/毫升抗体和20% 甘油(PBS) 在硝化纤维 (或功能化玻璃) 上使用喷墨微阵列检测幻灯片, 在相对湿度为 60% (每点 1.2 nL) 的情况下使用800和 #181; m 中心间距。确保幻灯片是固定在观察甲板根据一个角落 (这里, 左下角使用). </li>
		<li> 在室温下孵育 1 h 的斑点化验幻灯片, 相对湿度为 60%. </li>
		<li> 在化验幻灯片上夹住16隔间的滑动模块垫圈, 将其分成16口井。通过添加80和 #181 的 PBS, 冲洗三次幻灯片; 在每个井中都有 0.1% Tween-20 (PBST), 每次在摇床上晃动 450 rpm, 每一次都有5分钟. </li>
		<li> 添加80和 #181; L 对每个井的堵塞解决方案, 并在 450 rpm 时抖动1小时. </li>
		<li> 取下垫片, 用氮气流干燥化验滑块. </li>
		<li> 在观察甲板上固定一张传送幻灯片, 并将幻灯片的左下角按下左下角的探测甲板。喷墨点一组对准标记 (聚苯乙烯微珠的解决方案) 与驾驶室的布局相同. </li>
		<li> 让校准标记为干燥。翻转转移幻灯片, 并把它放回观察员甲板上, 在左下角固定. </li>
		<li> 准备民建联的解决方案, 包括20和 #181; g/毫升抗体, 20% 甘油和 1% BSA. </li>
		<li> 使用监视器和 #39 的摄像头, 拍摄对齐标记的图片。把这张图片作为一个基准来实现, 并得到喷墨观测器的图像识别系统, 以确定最左上标记。使用它的坐标作为第一个点的民建联阵列。点 8 nL 每液滴与中心间距800和 #181; m. </li>
	</ol> </li> <li> 双传输方法 ( 图 1b ) <ol> <li> 将传输幻灯片1放在底部左下角的喷墨检测甲板上。包含400和 #181 的现场 cab 解决方案; PBS 中的 g/毫升抗体, 20% 甘油和 1% BSA, 在相对湿度为60% 的喷墨微阵列检测 (每个点 0.4 nL, 直径为200和 #181) 的幻灯片上。中心间距为400和 #181; m. </li>
		<li> 使用一台 snap 设备 ( 图 2 ) 将转印的小水滴转到化验幻灯片上, 用一个硝化棉涂层 (或功能化的玻璃) 检测幻灯片来对传送幻灯片进行捕捉。
		<ol> <li> 将 snap 设备一侧的所有六按钮按90度打开, 并将所有活塞在打开的位置上锁定。将传送滑块插入其插座中的滑动架上, 并将所修剪的拐角朝向固定的弹簧针。打开第一组三按钮, 以释放活塞与金色的弹簧针, 并确保幻灯片是推对齐针. </li>
			<li> 在4弹簧脚上倒置的装置上插入一个硝化纤维素涂层的滑动器。打开第二组三按钮, 释放活塞与银针, 并确保幻灯片是推对齐针脚. </li>
			<li> 通过将顶部壳体放置在滑动架上, 使用对齐柱和孔进行精确定位, 从而关闭 snap 装置. </li>
			<li> 将闭合的 snap 装置插入其保持架中, 并将关闭标签完全向下推送, 以便在应用适当压力时面对面地进行微阵列。保持关闭1分钟. </li>
		</ol> </li> <li> 分离幻灯片。在室温下孵育 1 h 的化验幻灯片, 相对湿度为60%。如步骤 1.1.3-1.1.5 所述, 清洗、阻挡和干燥化验幻灯片. </li>
		<li> 在喷墨探测甲板上放置另一个传送幻灯片, 然后按下左下角。斑点民建联解决方案包含50或100和 #181; 抗体的 g/毫升 (见表 1), 20% 甘油, 和 1% BSA 在 PBS。确保每个点都是 0.8 nL, 点之间的中心间距是400和 #181; m. </li>
		<li> 存储检测和转移幻灯片。在含有干燥剂的密闭袋中密封化验和转移幻灯片, 并放入-20 和 #176; C 冷冻. </li>
	</ol> </li> </ol> 2。多路免疫与 snap 芯片 <ol> <li> 从冰箱中检索化验幻灯片。把袋子密封30分钟, 直到幻灯片到室温. </li>
	<li> 在含有 0.05% Tween-20 的 PBS 中通过峰值蛋白质制备7点系列稀释样品溶液. 
	注: 在这里, 使用5倍的稀释因子。表1列出了每个蛋白质的起始浓度. </li>
	<li> 根据需要准备示例解决方案。例如, 在 PBST 缓冲液中稀释人血清4倍. </li>
	<li> 在化验幻灯片上夹住一个16室垫片. </li>
	<li> 用7蛋白稀释溶液和无蛋白质的 PBST 缓冲液, 用移填充一列8口井。在同一张幻灯片的其他8口井中抽取样品溶液. 
	注:80 和 #181; L 解决方案可以适合每一个井。必要时可以使用更多的幻灯片来测量其他示例. </li>
	<li> 在室温下以 450 rpm 将样品孵化为1小时, 或在4和 #176 过夜; c. 在振动筛上用 PBST 冲洗三次, 每次450分钟。取下垫片, 在氮气流下烘干滑块. </li>
	<li> 从冷冻库中取出带有轻的传输幻灯片。在室温下将袋子密封30分钟。然后, 在关闭的腔室中孵育幻灯片 ( 例如 空提示框), 其中包含60% 湿度稳定珠, 用于20分钟的补液. </li>
	<li> 使用 snap 设备 ( 图 2 ) 将传输幻灯片与分析幻灯片对齐, 以传输 "民建联" 液滴。有关 snap 设备的操作, 请参见1.2.2 节. </li>
	<li> 将幻灯片分开。在包含60% 湿度稳定珠的密闭腔中孵育 1 h. 用16室垫片夹住化验幻灯片, 用 PBST 在 450 rpm、5分钟的振动筛上冲洗4次幻灯片. </li>
	<li> 吸管80和 #181; 包含2.5 和 #181 的溶液的 L; 亲和荧光在 PBS 中的每一个井。在 450 rpm 的振动筛上孵育20分钟. </li>
	<li> 用 PBST 冲洗幻灯片3次, 在 450 rpm 的情况下用蒸馏水在振动筛上清洗一次。取下垫片, 用氮气将滑块擦干. </li>
</ol> 3。幻灯片扫描和数据分析 <ol> <li> 使用 635 nm 激光扫描带有荧光微阵列扫描的检测幻灯片. </li>
	<li> 使用分析软件 (如阵列 pro 分析器) 16 提取每个点的净强度. </li>
	<li> 使用统计软件计算每个蛋白质的检测限度 (LOD), 并确定样本中的蛋白质浓度. 
	注意: LOD 被定义为标准 cu 的 Y 截距rve 增加了三倍的标准偏差三独立的化验. </li>
</ol>

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麦吉尔大学在这项工作的一些方面提出了专利申请, 杨惠妍和大卫. 容克作为发明者。

在这项工作中, 我们提出了一个 snap 芯片技术, 使交叉免费复免疫广泛适用于研究人员的基本实验设置。与现有的抗体微阵列不同, 不需要 end-users。单和双传输方法都证明, 双转移提供了优越的对准精度下降到〜40μ m 为98% 点, 与最大的失调63µm14。开发了一种新颖的 snap 装置, 可以方便地将两张幻灯片保持一致的压力, 使这项技术能够被研究人员使用。可靠的试剂传递不仅在硝化棉?...

一个由多种蛋白质组成的生物标志物的面板比单一的标志物在诊断复杂疾病如癌症1,2中提供更高的灵敏度和特异性。酶联免疫吸附试验 (ELISA) 是在临床实验室中使用的金标准技术, 在低 pg/毫升的等离子体中达到了检测的极限, 但每个化验值限制为一个目标3,4,5。抗体微阵列已开发, 以容纳数以千计的微型检测并行进行的单一显微镜幻灯片6,7,8。但是, 这种方法的复用能力受到试剂驱动交叉的限制, 这是由轻混合的应用引起的, 并且越来越多的目标9,10,11. 解放军et al。指出, 复合夹层分析结果的脆弱性是 4 n (n-1), 其中 n 是目标12的数目。
为了减少抗体微阵列中的交叉, 在我们的实验室中开发了抗体定位基因芯片 (ACM), 用于多重夹层试验12。捕获抗体 (cab) 被发现在基板上的微阵列的观察员。在表面上应用阻断样品后, 在与驾驶室抗原复合物相同的斑点上发现单个轻。所有交叉方案之间的抗体和抗原可以减轻与 ACM, 并限制检测在 pg/毫升已经实现。然而, 该检测协议要求在实验过程中使用一种高精度的 on-site 微阵列探测器来准备和识别轻, 这是昂贵和耗时的, 这限制了该技术的广泛应用其他实验室。一个手持式 ACM, 命名的 snap 芯片已开发为无交叉和无检举的复合夹层免疫13,14,15。驾驶室和轻是 pre-spotted 到一个化验幻灯片和转移幻灯片分别在微阵列格式和存储。在分析过程中, 通过简单地将两个芯片合在一起, 将检索到幻灯片, 并将轻的微阵列集体转移到化验幻灯片上。一种用于可靠的试剂转移的 snap 装置。具有相对较大的抗体结合能力的硝化纤维素切片被用作化验幻灯片, 以吸收液滴, 从而促进试剂的转移, 但是, 幻灯片比普通的玻片和微阵列更昂贵与非透明幻灯片兼容的扫描仪需要进行信号采集。
在这项工作中, 我们演示了执行一个多路三明治免疫分析与快照芯片的协议。为更方便、可靠的试剂从 microarray-to-microarray 中转移, 研制了一种新型的抓拍装置。重要的是, 在这里, 我们已经建立了试剂传输方法到常规玻璃幻灯片与快照芯片。1024斑点成功地被转移了并且被排列了到玻璃幻灯片, 极大地扩展这技术的用途在多数实验室。

<table><tbody><tr><td>Phosphate buffered saline tablet</td><td>Fisher Scientific</td><td>5246501EA</td><td></td></tr><tr><td>Streptavidin-conjugated Cy5</td><td>Rockland</td><td>s000-06</td><td></td></tr><tr><td>Tween-20</td><td>Sigma-Aldrich</td><td>p1379</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Jackson ImmunoResearch Laboratories, Inc</td><td>001-000-162</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G5516</td><td></td></tr><tr><td>Blocking solution: BSA-free StabilGuard Choice Microarray Stabilizer</td><td>SurModics, Inc</td><td>SG02</td><td></td></tr><tr><td>Nitrocellulose coated slides</td><td>Grace Bio-Laboratories, Inc</td><td>305116</td><td></td></tr><tr><td>Aminosilane coated slides</td><td>Schott North America</td><td>1064875</td><td></td></tr><tr><td>Snap Device</td><td>Parallex BioAssays Inc.</td><td>PBA-SD01</td><td></td></tr><tr><td>Inkjet microarray spotter</td><td>GeSiM</td><td>Nanoplotter 2.0</td><td></td></tr><tr><td>Slide module gasket</td><td>Grace Bio-Laboratories, Inc</td><td>204862</td><td></td></tr><tr><td>Humidity Stabilization Beads</td><td>Parallex BioAssays Inc.</td><td>PBA-HU60</td><td></td></tr><tr><td>Array-Pro Analyzer software</td><td>Media Cybernetics</td><td>Version 4.5</td><td></td></tr><tr><td>Fluorescence microarray scanner</td><td>Agilent</td><td>SureScan Microarray Scanner</td><td></td></tr><tr><td>Biostatistics software</td><td>GraphPad Software</td><td>GraphPad Prism 6</td><td></td></tr><tr><td>Endoglin capture antibody</td><td>R&amp;amp;D Systems</td><td>MAB10972</td><td></td></tr><tr><td>Endoglin protein</td><td>R&amp;amp;D Systems</td><td>1097-EN</td><td></td></tr><tr><td>Endoglin detection antibody</td><td>R&amp;amp;D Systems</td><td>BAF1097</td><td></td></tr><tr><td>IL-6&lt;sup&gt;a (see Table 1)&lt;/sup&gt;</td><td>R&amp;amp;D Systems</td><td></td><td></td></tr><tr><td>IL-6&lt;sup&gt;b (see Table 1)&lt;/sup&gt;</td><td>Invitrogen</td><td></td><td></td></tr></tbody></table>

snap chip for cross-reactivity-free and spotter-free multiplexed sandwich immunoassays

多路复用蛋白分析显示, 与单一蛋白相比, 诊断灵敏度和准确度均较高。抗体微阵列允许在单个芯片上同时进行数以千计的微型免疫。三明治化验格式通过检测每个目标的两个抗体, 提高了检测的特异性, 但在试剂之间交叉, 从而限制了它们的复用能力。抗体定位微阵列 (ACM) 已发展为无交叉多路复用蛋白质检测, 但需要一个昂贵的观察员 on-site 芯片制造过程中的化验。在这项工作中, 我们展示了一个 snap 芯片技术, 通过简单地将两个芯片一起从 microarray-to-microarray 转移试剂, 因此在样品孵化和随后应用检测抗体 (轻) 时不需要任何的观测存储 pre-spotted 的幻灯片, 游离的幻灯片准备从化验执行。本文介绍了两种单片和双转印方法, 实现了双微阵列的精确对准, 并描述了这两个方法的滑动制作。结果表明, #60; 40 μ m 对准实现了双转移, 达到了625点/cm2的阵列密度。50-plexed 免疫分析已经进行了演示芯片的可用性, 在多路复用蛋白质分析仪。检测35种蛋白质的限度在 pg/毫升的范围内。

单和双传输方法的检测过程如图 1所示。在单转移, 驾驶室被发现直接在化验幻灯片和轻被转移到化验幻灯片后使用的一个镜像模式的驾驶室 (图 1a)。只有一个转移过程是必需的, 但这种方法是由两个微阵列之间的失调, 主要是由于在幻灯片和喷墨龙门之间的角度失调 (图 2) 造成的。解决这一难题的一种方?...

Watch this Scientific Journal Video about Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays at JoVE.com

Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays

我们展示了一个快照技术, 执行无交叉的多路复用三明治免疫简单地捕捉两个幻灯片。一种用于可靠地从 microarray-to-microarray 中转移试剂的 snap 装置。该 snap 芯片可用于任何生化反应需要定位不同的试剂没有交叉污染。

无交叉反应和无免疫多路复用夹芯芯片

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands ...

snap-chip-for-cross-reactivity-free-spotter-free-multiplexed-sandwich

Research

JoVE Journal

Bioengineering

6.5K 次观看.  McGill University and Génome Québec Innovation Centre. 我们展示了一个快照技术, 执行无交叉的多路复用三明治免疫简单地捕捉两个幻灯片。一种用于可靠地从 microarray-to-microarray 中转移试剂的 snap 装置。该 snap 芯片可用于任何生化反应需要定位不同的试剂没有交叉污染。

视频: Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays

Multiplexed Fluorescent Microarray for Human Salivary Protein Analysis Using Polymer Microspheres and Fiber-optic Bundles

Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array technology employed combines the advantages of microsphere-based suspension array fabrication with the use of fluorescence microscopy. As described in the video protocol, commercially available 4.5 &#956;m polymer microspheres were encoded into seven different types, differentiated by the concentration of two fluorescent dyes physically trapped inside the microspheres. The encoded microspheres containing surface carboxyl groups were modified with monoclonal capture antibodies through EDC/NHS coupling chemistry. To assemble the protein microarray, the different types of encoded and functionalized microspheres were mixed and randomly deposited in 4.5 &#956;m microwells, which were chemically etched at the proximal end of a fiber-optic bundle. The fiber-optic bundle was used as both a carrier and for imaging the microspheres. Once assembled, the microarray was used to capture proteins in the saliva supernatant collected from the clinic. The detection was based on a sandwich immunoassay using a mixture of biotinylated detection antibodies for different analytes with a streptavidin-conjugated fluorescent probe, R-phycoerythrin. The microarray was imaged by fluorescence microscopy in three different channels, two for microsphere registration and one for the assay signal. The fluorescence micrographs were then decoded and analyzed using a homemade algorithm in MATLAB.

We describe a procedure for profiling salivary proteins using multiplexed microsphere-based antibody arrays. Monoclonal antibodies were covalently linked to fluorescent dye-encoded 4.5 &#x03BC;m polymer microspheres using carbodiimide chemistry. The modified microspheres were deposited in fiber-optic microwells to measure protein levels in saliva using fluorescence sandwich immunoassays.

复用荧光微阵列人类唾液蛋白质分析用高分子微球和光纤束

Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array ...

科研

化学

Fabricating a UV-Vis and Raman Spectroscopy Immunoassay Platform

Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, a large infrastructure of immunoassay instruments and materials can be found. For example, 96- and 384-well polystyrene plates are available commercially and have a standard design to accommodate ultraviolet-visible (UV-Vis) spectroscopy machines from various manufacturers. In addition, a wide variety of immunoglobulins, detection tags, and blocking agents for customized immunoassay designs such as enzyme-linked immunosorbent assays (ELISA) are available.
Despite the existing infrastructure, standard ELISA kits do not meet all research needs, requiring individualized immunoassay development, which can be expensive and time-consuming. For example, ELISA kits have low multiplexing (detection of more than one analyte at a time) capabilities as they usually depend on fluorescence or colorimetric methods for detection. Colorimetric and fluorescent-based analyses have limited multiplexing capabilities due to broad spectral peaks. In contrast, Raman spectroscopy-based methods have a much greater capability for multiplexing due to narrow emission peaks. Another advantage of Raman spectroscopy is that Raman reporters experience significantly less photobleaching than fluorescent tags1. Despite the advantages that Raman reporters have over fluorescent and colorimetric tags, protocols to fabricate Raman-based immunoassays are limited. The purpose of this paper is to provide a protocol to prepare functionalized probes to use in conjunction with polystyrene plates for direct detection of analytes by UV-Vis analysis and Raman spectroscopy. This protocol will allow researchers to take a do-it-yourself approach for future multi-analyte detection while capitalizing on pre-established infrastructure.

Nanoparticle-based optical probes have been designed as a vehicle for detecting antigens using Raman and UV-Vis spectroscopy. Here we describe a protocol for preparing such probes for a UV-Vis/Raman spectroscopy immunoassay in such a way to incorporate future multiplexing capabilities.

制造的UV-Vis和拉曼光谱免疫平台

Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, ...

生物化学

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has relevant health and economic consequences. Many cyanobacterial strains are toxin producers, and only a few cells are necessary to induce irreparable damage to the environment. Therefore, water-body authorities and administrations require rapid and efficient early-warning systems providing reliable data to support their preventive or curative decisions. This manuscript reports an experimental protocol for the in-field detection of toxin-producing cyanobacterial strains by using an antibody microarray chip with 17 antibodies (Abs) with taxonomic resolution (CYANOCHIP). Here, a multiplex fluorescent sandwich microarray immunoassay (FSMI) for the simultaneous monitoring of 17 cyanobacterial strains frequently found blooming in freshwater ecosystems, some of them toxin producers, is described. A microarray with multiple identical replicates (up to 24) of the CYANOCHIP was printed onto a single microscope slide to simultaneously test a similar number of samples. Liquid samples can be tested either by direct incubation with the antibodies (Abs) or after cell concentration by filtration through a 1- to 3-&#956;m filter. Solid samples, such as sediments and ground rocks, are first homogenized and dispersed by a hand-held ultrasonicator in an incubation buffer. They are then filtered (5 - 20 &#956;m) to remove the coarse material, and the filtrate is incubated with Abs. Immunoreactions are revealed by a final incubation with a mixture of the 17 fluorescence-labeled Abs and are read by a portable fluorescence detector. The whole process takes around 3 h, most of it corresponding to two 1-h periods of incubation. The output is an image, where bright spots correspond to the positive detection of cyanobacterial markers.

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

The presence of cyanobacterial toxins in fresh water reservoirs for human consumption is a major concern for water management authorities. To evaluate the risk of water contamination, this article describes an protocol for the in-field detection of cyanobacterial strains in liquid and solid samples by using an antibody microarray chip.

用于检测实验方案<em&gt;蓝藻</em&gt;与抗体基因芯片液体和固体样品

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has ...

环境科学

Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different downstream patient-to-patient transmission potentials, and require very different patient treatments. Thus, recent Zika outbreaks make it urgent to develop tools that rapidly discriminate these viruses in patients and trapped mosquitoes, to select the correct patient treatment, and to understand and manage their epidemiology in real time.
Unfortunately, current diagnostic tests, including those receiving 2016 emergency use authorizations and fast-track status, detect viral RNA by reverse transcription polymerase chain reaction (RT-PCR), which requires instrumentation, trained users, and considerable sample preparation. Thus, they must be sent to &#34;approved&#34; reference laboratories, requiring time. Indeed, in August 2016, the Center for Disease Control (CDC) was asking pregnant women who had been bitten by a mosquito and developed a Zika-indicating rash to wait an unacceptable 2 to 4 weeks before learning whether they were infected. We very much need tests that can be done on site, with few resources, and by trained but not necessarily licensed personnel.
This video demonstrates an assay that meets these specifications, working with urine or serum (for patients) or crushed mosquito carcasses (for environmental surveillance), all without much sample preparation. Mosquito carcasses are captured on paper carrying quaternary ammonium groups (Q-paper) followed by ammonia treatment to manage biohazards. These are then directly, without RNA isolation, put into assay tubes containing freeze-dried reagents that need no chain of refrigeration. A modified form of reverse transcription loop-mediated isothermal amplification with target-specific fluorescently tagged displaceable probes produces readout, in 30 min, as a three-color fluorescence signal. This is visualized with a handheld, battery-powered device with an orange filter. Forward contamination is prevented with sealed tubes, and the use of thermolabile uracil DNA glycosylase (UDG) in the presence of dUTP in the amplification mixture.

Current multiplexed diagnostics to detect Zika, chikungunya, and dengue viruses require complex sample preparation and expensive instrumentation, and are difficult to use in low resource environments. We show a diagnostic that uses isothermal amplification with target-specific strand displaceable probes to detect and differentiate these viruses with high sensitivity and specificity.

基于复用等温放大诊断平台检测 Zika、基孔肯亚和登革热1

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different ...

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

一个微流控芯片单细胞的多功能化学分析

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

生物工程

Multiplexed Barcoding Image Analysis for Immunoprofiling and Spatial Mapping Characterization in the Single-Cell Analysis of Paraffin Tissue Samples

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is an effective methodology for tumor evaluation that improves the understanding of the tumor microenvironment. The visualization of protein expression in formalin-fixed, paraffin-embedded&#160;tissues is achieved when a specific fluorophore is annealed to an antibody-bound barcode via complementary oligonucleotides and then sample imaging is performed; indeed, this method allows for the use of customizable panels of more than 40 antibodies in a single tissue staining reaction. This method is compatible with fresh frozen tissue, formalin-fixed, paraffin-embedded tissue, cultured cells, and peripheral blood mononuclear cells, meaning that researchers can use this technology to view a variety of sample types at single-cell resolution. This method starts with a manual staining and fixing protocol, and all the antibody barcodes are applied using an antibody cocktail. The staining fluidics instrument is fully automated and performs iterative cycles of labeling, imaging, and removing spectrally distinct fluorophores until all the biomarkers have been imaged using a standard fluorescence microscope. The images are then collected and compiled across all the imaging cycles to achieve single-cell resolution for all the markers. The single-step staining and gentle fluorophore removal not only allow for highly multiplexed biomarker analysis but also preserve the sample for additional downstream analysis if desired (e.g., hematoxylin and eosin staining). Furthermore, the image analysis software enables image processing-drift compensation, background subtraction, cell segmentation, and clustering-as well as the visualization and analysis of the images and cell phenotypes for the generation of spatial network maps. In summary, this technology employs a computerized microfluidics system and fluorescence microscope to iteratively hybridize, image, and strip fluorescently labeled DNA probes that are complementary to tissue-bound, oligonucleotide-conjugated antibodies.

Multiplexed barcoding image analysis has recently improved the characterization of the tumor microenvironment, permitting comprehensive studies of cell composition, functional state, and cell-cell interactions. Herein, we describe a staining and imaging protocol using the barcoding of oligonucleotide-conjugated antibodies and cycle imaging, which allows for the use of a high-dimensional image&#160;analysis technique.

多重条形码图像分析，用于石蜡组织样品单细胞分析中的免疫分析和空间映射表征

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is ...

癌症研究

Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and infections using cost effective, high-throughput approaches would be indispensable. This manuscript describes the development and analysis of a bead-based multiplex immunoassay capable of measuring the presence of antibodies in human saliva to multiple pathogens simultaneously. Saliva is particularly attractive in this application because it is noninvasive, cheaper and easier to collect than serum. Antigens from environmental pathogens were coupled to carboxylated microspheres (beads) and used to measure antibodies in very small volumes of human saliva samples using a bead-based, solution-phase assay. Beads were coupled with antigens from Campylobacter jejuni, Helicobacter pylori, Toxoplasma gondii, noroviruses (G I.1 and G II.4) and hepatitis A virus. To ensure that the antigens were sufficiently coupled to the beads, coupling was confirmed using species-specific, animal-derived primary capture antibodies, followed by incubation with biotinylated anti-species secondary detection antibodies and streptavidin-R-phycoerythrin reporter (SAPE). As a control to measure non-specific binding, one bead set was treated identically to the others except it was not coupled to any antigen. The antigen-coupled and control beads were then incubated with prospectively-collected human saliva samples, measured on a high throughput analyzer based on the principles of flow cytometry, and the presence of antibodies to each antigen was measured in Median Fluorescence Intensity units (MFI). This multiplex immunoassay has a number of advantages, including more data with less sample; reduced costs and labor; and the ability to customize the assay to many targets of interest. Results indicate that the salivary multiplex immunoassay may be capable of identifying previous exposures and infections, which can be especially useful in surveillance studies involving large human populations.

In the current climate of scarce resources, new technologies are emerging that allow researchers to conduct studies cheaper, faster and with more precision. Here we describe the development of a bead-based salivary antibody multiplex immunoassay to measure human exposure to multiple environmental pathogens simultaneously.

制定唾液抗体多重免疫测定来测量人体暴露于环境中的病原体

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and ...

免疫与感染

Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life science research and clinical diagnostics. However, ELISA has limitations. It is typically performed in a 96-well microplate, and the wells are coated with capture antibody, requiring a relatively large amount of sample to capture an antigen of interest . The large surface area of the wells and the hydrophobic binding of capture antibody can also lead to non-specific binding and increased background. Additionally, most ELISAs rely upon enzyme-mediated amplification of signal in order to achieve reasonable sensitivity. Such amplification is not always linear and can thus skew results.
In the past 15 years, a new technology has emerged that offers the benefits of the ELISA, but also enables higher throughput, increased flexibility, reduced sample volume, and lower cost, with a similar workflow 1, 2. Luminex xMAP Technology is a microsphere (bead) array platform enabling both monoplex and multiplex assays that can be applied to both protein and nucleic acid applications 3-5. The beads have the capture antibody covalently immobilized on a smaller surface area, requiring less capture antibody and smaller sample volumes, compared to ELISA, and non-specific binding is significantly reduced. Smaller sample volumes are important when working with limiting samples such as cerebrospinal fluid, synovial fluid, etc. 6. Multiplexing the assay further reduces sample volume requirements, enabling multiple results from a single sample.
Recent improvements by Luminex include: the new MAGPIX system, a smaller, less expensive, easier-to-use analyzer; Low-Concentration Magnetic MagPlex Microspheres which eliminate the need for expensive filter plates and come in a working concentration better suited for assay development and low-throughput applications; and the xMAP Antibody Coupling (AbC) Kit, which includes a protocol, reagents, and consumables necessary for coupling beads to the capture antibody of interest. (See Materials section for a detailed list of kit contents.)
In this experiment, we convert a pre-optimized ELISA assay for TNF-alpha cytokine to the xMAP platform and compare the performance of the two methods 7-11. TNF-alpha is a biomarker used in the measurement of inflammatory responses in patients with autoimmune disorders.
We begin by coupling four candidate capture antibodies to four different microsphere sets or regions. When mixed together, these four sets allow for the simultaneous testing of all four candidates with four separate detection antibodies to determine the best antibody pair, saving reagents, sample and time. Two xMAP assays are then constructed with the two most optimal antibody pairs and their performance is compared to that of the original ELISA assay in regards to signal strength, dynamic range, and sensitivity.

An ELISA can be easily converted to a Luminex xMAP assay and, through the benefits of multiplexing, several antibodies can be screened simultaneously to identify an optimum antibody pair, resulting in increased sensitivity and dynamic range, while reducing assay cost.

转换到一个点阵仪XMAP含量，使用多重抗体筛查方法的捕获ELISA

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life ...

生物学

血清中 疏螺旋体特异性 IgG 和 IgM 的多重免疫检测

Simultaneous Detection of Different Antibody Classes in a Multiplexed Serological Test

To monitor the progression of infectious diseases, it is useful to assess immunoreactivity against various antigenic determinants, and measure different antibody isotypes because they appear at different stages of the host immune response. With Lyme borreliosis, the pathogenic agent can be one of the multiple members of the Borrelia species. Therefore, correct sample classification requires evaluating the immunoreactivity against different antigens of different Borrelia species. Additionally, anti-pathogen IgG and IgM responses can have different elicitation time courses during disease progression. Here we demonstrate the development of a two-reporter multiplex immunoassay that has utility in identifying Borrelia-specific immune response in human serum samples by simultaneously evaluating both IgG and IgM immunoreactivity against different bacterial antigens in the same reaction well. This dual-reporter approach retains the analytical performance of single-reporter methods while conserving time and resources and reducing sample size requirements. This assay allows essentially double the serological information to be generated from a blood sample in half the time.

作者聚焦:扩大用于莱姆疏螺旋体病诊断和病原体研究的多重免疫检测的范围 

A three-channel dual-reporter fluorescence flow analysis system was used to develop a bead-based multiplex immunoassay that simultaneously evaluates serum samples for IgG and IgM elicited against multiple antigens of different Borrelia species that cause Lyme borreliosis in Europe and North America.

在多重血清学检测中同时检测不同抗体类别

To monitor the progression of infectious diseases, it is useful to assess immunoreactivity against various antigenic determinants, and measure different ...

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摘要

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在多重血清学检测中同时检测不同抗体类别

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